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Establishment of recombinant cannabinoid receptor assays and characterization of several natural and synthetic ligands

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Abstract

Cannabinoid receptors (CBR) are important drug targets for the treatment of various inflammatory, metabolic and neurological diseases. Therefore, sensitive test systems for the assessment of ligands are needed. In this study, a steady-state GTPase assay for human CBR subtypes 1 and 2 was developed to characterize the pharmacological property of ligands at a very proximal point of the signal transduction cascade. Establishing these in vitro test sytems, we studied cell or tissue membranes heterogenously or endogenously expressing CBR, such as CBR-infected Human Embryonic Kidney (HEK) 293 cells, rat cerebellum and spleen cells. The lack of effects in the GTPase assay and in [35S]GTPγS binding experiments in these expression system, directed us to use Spodoptera frugiperda (Sf9) cells. Co-expressing CBR, different Gα-subunits, Gβγ heterodimer, and RGS (Regulator of G-protein signaling)-proteins in Sf9 cell membranes greatly improved the sensitivity of the assay, with highest GTPase activation in the CBR + Gαi2 + Gβ1γ2 + RGS4 system. We examined exogenous and endogenous standard ligands as well as secondary metabolites as Δ9-tetrahydrocannabinol (Δ9-THC), dodeca-2E,4E-dienoic acid isobutylamide, an alkylamide from Echinacea purpurea, and an E. purpurea hexane extract according their agonistic and antagonistic properties. The suitability of the assay for screening procedures was also proven by detecting the activity of Δ9-THC in a matrix of other less active compounds (Δ9-THC-free Cannabis sativa extract). In conclusion, we have developed highly sensitive test systems for the analysis of CBR ligands.

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Acknowledgments

We thank G. Wilberg (Department of Pharmacology and Toxicology, University of Regensburg) and K. Fisch (Department of Pharmaceutical Biology, University of Regensburg) for technical assistance; Dr. K. Wenzel-Seifert (Department of Pharmacology and Toxicology, University of Regensburg) for her professional support concerning cell culture, transfection, and cell imaging; Dr. D. Schnell (Department of Pharmacology and Toxicology, University of Regensburg) for his help to synthesize [γ-32P]GTP and O. Bosch for his friendly support with the animal studies. Thanks are also due to Professor Dr. J. Schlossmann (Department of Pharmacology and Toxicology, University of Regensburg) for providing infrastructure and the reviewers for their constructive critique. Research support given by the Deutsche Forschungsgemeinschaft (Graduiertenkolleg 760 “Medicinal Chemistry”, University of Regensburg) is gratefully acknowledged.

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Authors and Affiliations

  1. Department of Pharmaceutical Biology, Institute of Pharmacy, University of Regensburg, 93053, Regensburg, Germany

    Sarah Geiger, Kathrin Nickl & Jörg Heilmann

  2. Department of Pharmacology and Toxicology, Institute of Pharmacy, University of Regensburg, Regensburg, Germany

    Erich H. Schneider

  3. Institute of Pharmacology, Medical School of Hannover, Hannover, Germany

    Roland Seifert

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  1. Sarah Geiger
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Correspondence to Jörg Heilmann.

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Geiger, S., Nickl, K., Schneider, E.H. et al. Establishment of recombinant cannabinoid receptor assays and characterization of several natural and synthetic ligands. Naunyn-Schmied Arch Pharmacol 382, 177–191 (2010). https://doi.org/10.1007/s00210-010-0534-5

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